CN109576273A - A set of tumor-marker molecular nucleic acid aptamers and application based on CRISPR/Cas9 - Google Patents
A set of tumor-marker molecular nucleic acid aptamers and application based on CRISPR/Cas9 Download PDFInfo
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Abstract
The present invention discloses a set of tumor-marker molecular nucleic acid aptamers and application based on CRISPR/Cas9, belongs to genetic engineering, immunology and oncology technical field.The aptamers are one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID.Aptamer of the present invention is obtained using full Cell depletion Cell-SELEX technology screening, molecular weight is smaller, it is readily synthesized and modifies, can high specific identification and the tumor markers molecule such as high-affinity combination ATG16L1, CSNK2, PPP1, in conjunction with other cells not occurrence features, non-immunogenicity stablizes easily modification, convenient for synthesis and saves.The tumor-marker molecular detection kit and reagent paper prepared with the aptamers is easy to optimize to tumor-marker Molecular Detection high sensitivity.It is applied in diagnosing tumor, tumor prognosis and the assessment of monitoring, diagnosing tumor.
Description
Technical field
The invention belongs to genetic engineering, immunology and oncology technical fields, in particular to a set of to be based on CRISPR/Cas9
Tumor-marker molecular nucleic acid aptamers and application.
Background technique
Autophagy GAP-associated protein GAP of the autophagy system by some evolution is confirmed in research from saccharomycete to mammal
(ATG) stringent regulation.Autophagy starts from BECN1/Beclin 1 and Group III phosphatidyl-inositol 3-kinase (PIK3C3), it has
Help phagocytic vacuole and absorbs other ATG albumen formation nucleus.In amplification, Cytoplasmic inclusions are directly inhaled by double film phagocytic vacuoles
It receives, then generates complete autophagosome；Autophagosome and lysosome fusion degradation other materials.Autophagy body maturation needs a series of
Ubiquitin sample is conjugated event, relates generally to LC3- phosphatidyl-ethanolamine (PE) and ATG12-ATG5-ATG16L1 conjugated system.These
Core ATG albumen, including ATG16L1 are all that autophagosome is indispensable.ATG16L1 interacts conjugation with ATG12-ATG5 and is formed
Dimer complex, target phagocyte and by serve as a kind of lipid E3 ubiquitin-like ligase promote MAP1LC3B/
1 light chain 3 of LC3B(microtubule-associated proteins b) is esterified, although function course of the ATG16L1 in autophagy process has good grounds,
It knows little about it to the active regulatory mechanism of ATG16L1.
CSNK2 is one highly conserved, composition, the serine-threonine kinase of wide expression, is one and includes
The tetramer holoenzyme (CSNK2A1/a and/or CSNK2A2/a') of 2 catalytic subunits and 2 supervision subunits (CSNK2B/
B) compound.The enzyme undertakes the phosphorylation more than 300 substrates and influences a series of bioprocess, including chromosome point
From spindle is formed, and Apoptosis is adjusted, and controls cell Proliferation and circadian rhythm.Recent research have also shown that aPPP1 is certainly
It bites and plays potential effect in regulation.
Protein phosphorylation can be reversed by phosphatase etc., such as PPP1, a kind of eukaryon serine/Soviet Union's ammonia generally expressed
Acid is the phosphoprotein phosphatase that can influence various kinds of cell function and process.The protein phosphatase enzyme family of PPP1 ownership includes 4 main
Subfamily: PPP1, PPP2CA, PPP2CB and PPP5.It is interesting that as CSNK2, PPP2(phosphoprotein phosphatase 2) promote in person
It bites BECN1 activity and BECN1 is changed in Ras-NIH 3T3 cell by induced conformational.
Aptamer is that one kind can have and anti-with the DNA or RNA sequence in conjunction with target molecule high specific high-affinity
The similar effect of body, commonly referred to as " chemical antibody ".In recent years, with the continuous development of aptamer screening technique, more
It is found come more tumor associated nucleic acid aptamers sequences by people.However, the relevant aptamer of these tumours is usually
It is only capable of identifying a kind of tumour cell or a kind of tumour, largely limits the application of aptamer.
CRISPR/Cas9 technology is the object of numerous scholar researchs and application as hot technology in recent years,
CRISPR/Cas9 technology carries out targeting knockout (Knock out) to target cell target gene or knocks in (Knock in) Duo Shiyong
In gene therapy, constructing the further biological study of cell model progress with this mode, there are also to be developed.Currently, Cell-
It is the tumour cell progress for being directed to different types of unknown target spot that it is mostly, which to carry out aptamer screening, by SELEX.But different tumours are thin
There is otherness between born of the same parents, lack common target molecules.Therefore, it is constructed using CRISPR/Cas9 technology and stablizes expression purpose
Gene surely turn cell line, reduce the building time of cell model, reduce the construction cost of model, be those skilled in the art urgently
Problem to be solved.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of molecular weight is smaller, it is readily synthesized and modifies, propose a set of base
In the tumor-marker molecular nucleic acid aptamers of CRISPR/Cas9 and application.
Purpose to realize the present invention, the technical solution used are as follows: a set of tumor-marker molecular core based on CRISPR/Cas9
Sour aptamers, the aptamers are one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID.
Preferably, the aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or
The sequence that the homology of several sequences is 60% or more.
Preferably, the aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or
The sequence that several sequences are hybridized.
Preferably, the aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or
The sequence that several sequences are transcribed.
Preferably, the aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or
Several sequences optimize truncated sequence.
Preferably, several positions in the aptamers sequence be phosphorylated, methylate, amination, sulfhydrylation or
Isotopologue.
Preferably, it is combined with biotin in the aptamers sequence, digoxin, fluorescent material, nano luminescent material, gathers
Ethylene glycol, peptide fragment, albumen, folic acid or enzyme label.
Preferably, the tumor-marker molecular nucleic acid aptamers have and nucleotides sequence shown in NO:1 ~ 5 SEQ ID
One or several any sequence alterations in column at corresponding peptide nucleic acid.
The present invention also provides a set of, and the tumor-marker molecular nucleic acid aptamers based on CRISPR/Cas9 are preparing tumor-marker
The application of molecular detection kit or detection reagent paper.
Present invention substantive distinguishing features outstanding and significant progress are:
Aptamer of the present invention using full bacterium abatement Cell-SELEX technology screening and obtain, molecular weight is smaller, be readily synthesized and
Modification, can high specific identification and high-affinity combination ATG16L1, CSNK2, PPP1, with other targets not occurrence features
In conjunction with non-immunogenicity stablizes easily modification, convenient for synthesis and saves, can be used for detecting ATG16L1, CSNK2, PPP1, and have
The prevention of effect ground and control ATG16L1, CSNK2, PPP1 infection are of great significance.It is constructed and is stablized using CRISPR/Cas9 technology
Expression target gene surely turns cell line, can greatly reduce the building time of cell model, reduce the construction cost of model, energy
Solve the problems, such as living cells carry out aptamers screening when target molecules it is unknown, using the aptamers preparation ATG16L1,
CSNK2, PPP1 detection kit and reagent paper are high to ATG16L1, CSNK2, PPP1 detection sensitivity, and are easy to optimize.Wherein,
The present invention is only by taking PPP1 as an example.
Detailed description of the invention
Fig. 1 is PCR annealing temperature optimization figure.
3% agarose gel electrophoresis shows the PCR amplification effect for using library at different temperatures as template, swimming lane 1=
47.2 °C, 2=47.3 °C of swimming lane, 3=47.6 °C of swimming lane, 4=48.1 °C of swimming lane, 5=48.8 °C of swimming lane, 6=49.5 °C of swimming lane,
7=50.2 °C of swimming lane, 8=50.9 °C of swimming lane, 9=51.6 °C of swimming lane, 10=52.2 °C of swimming lane, 11=52.6 °C of swimming lane, swimming lane
The visible swimming lane 6 of 12=52.8 °C of swimming lane bp DNA ladder of M=20 marker has most bright band, therefore selects swimming
(49.5 °C) of 6 temperature of road are used as optimal annealing temperature.
Fig. 2 is the 19th wheel screening product sequencing comparison result figure according to the similitude in high conserved region domain in DNA sequence dna, will be surveyed
Sequence result is divided into five families.
Fig. 3 is candidate aptamers and 293T-PPP1 cell combination fluorescence imaging result (400x).
Fig. 4 is candidate aptamers and 293T cell combination fluorescence imaging result (400x).
Fig. 5 is influence of the pancreatin to PPP1-4 binding ability as pancreatin prolongs the target cell 293T- PPP1 processing time
Long, fluorescence intensity of the PPP1-4 in conjunction with 293T- PPP1 is in gradually reducing tendency.
Fig. 6 is each aptamers truncated sequence situation streaming result in conjunction with target cell.
A. compared with former sequence, PPP1-4a and the PPP1-4b fluorescence intensity in conjunction with target cell 293T- PPP1 have a little
Weaken, and PPP1-4c can maintain binding ability similar with former sequence.
B. PPP1-4a, PPP1-4b and PPP1-4c cannot be in conjunction with negative cells 293T.
Specific embodiment
The present invention program is described in further detail below with reference to embodiment, following the description is merely to explain this hair
It is bright, its content is not defined.
Embodiment:
Tumor-marker molecular nucleic acid aptamers and application based on CRISPR/Cas9 of the invention, the present embodiment by taking PPP1 as an example,
Specific technical solution is as follows:
The selection of the library 1.DNA
In aptamers screening process, with the progress of PCR, screening product can be converted from specificity to non-specificity.Cause
This should select itself higher library of G/C base ratio when carrying out library selection, to reduce PCR stage non-specificity
The generation of product.The aptamers that the present invention is delivered with reference to Kang etc. screen library used in pertinent literature: 5 '-GAA TTC
AGT CGG ACA GCG-N45-GAT GGA CGA ATA TCG TCT CCC-3 ', library GC ratio reach 51.8%, have
Higher G/C ratio, and it is intermediate contain 45 randomized bases, not only ensure that the rich of library, but not because length is too long and
Lead to structural instability.
2. PCR annealing temperature optimizes
Therefore the generation that suitable annealing temperature can effectively reduce non-specific band need to carry out annealing temperature in PCR condition
Optimization.With reference to the PCR condition in primer Tm and document report, annealing temperature optimization is carried out using temperature gradient PCR method
Investigation.The specific steps are setting annealing temperature between 47 °C to 53 °C, the arrangement of temperature gradient is carried out automatically by PCR instrument
Setting.After being completed to PCR reaction, PCR product is subjected to 3% agarose gel electrophoresis and is shot, concrete outcome such as Fig. 1 institute
Show.The results show that at 49.5 °C of document report temperature, PCR has the brightest band, and without disperse and non-specific
Property amplification, therefore select the temperature as subsequent PCR annealing temperature, the suitable annealing temperature as PCR in this screening.
3. screening process
The 293T-PPP1 cell line for stablizing high expression people PPP1 gene is successfully constructed using CRISPR/Cas9 technology.Aptamers sieve
Select process is that duplicate, progressive process is taken turns one more, any one link occur mistake all will affect to the end as a result, tight
Entire the failure of an experiment is even resulted in when weight.Therefore, it under the premise of in accordance with basic Cell-SELEX screening step, needs to details
Control, to improve the screening efficiency of aptamers.It is specifically as follows:
(1) guarantee cell state
Whether aptamers screening is successful to have very close relationship with cell state.Cell state may will affect it when poor
The normal expression of surface protein, leads to the missing or change of expected aptamers target, to influence the progress of aptamers screening.This
Two groups of positive and reverse screening cells that invention is selected are both attached cells of 293T-PPP1 and 293T respectively.Before each screening,
It is both needed to guarantee two kinds of cell culture times within 24-48h, to may cause cell adherent not yet this is because incubation time is too short
Reach perfect condition, and the too long cell that may cause of incubation time is adherent excessively securely, the link for scraping off cell and collecting compared with
For difficulty.In addition, cell also should be at growing logarithmic growth phase the most vigorous, and maintain vigour greater than 90%.Cell viability
Judgement is mainly to carry out according to trypan blue negative staining method, when cell activity is preferable, will not still can under mirror by Trypan Blue
Observe the good cell of translucency；When cell is dyed to blue as the result is shown under the microscope, prompt cell state poor, very
To death.
(2) selection of temperature is screened
Temperature can usually generate certain influence to the combination of cell and DNA.Therefore, suitable temperature is selected to screen aptamers
Success or not plays important decisive action.The screening selection that the present invention is carried out carries out under 4 °C of environment, this is because with
The extension of time can generate endocytosis to DNA in (such as 37 °C) cell under hot environment, affect the rich of library to a certain extent
Fu Xing, and reduction appropriate screening temperature can effectively avoid this phenomenon.
(3) it is actively introduced counter-selection
To obtain the aptamers having compared with high specific as early as possible, it should counter-selection choosing be added before the 3rd wheel as far as possible.In the present invention,
Anti- screening step is added in front of each round just screens, can not will directly select the DNA of cell combination to collect simultaneously with counter-selection in this way
It puts into positive screening cell, without carrying out other washing steps, avoids screening while simplifying screening step
The partial loss in library.
(4) increase of screening pressure
With the increase of number of screening round, it to be gradually increased screening pressure, can just effectively improve screening efficiency and success rate.Screening pressure
The increase of power is mainly reflected in wash time, washing times, incubation time etc..It specifically, is exactly with number of screening round
Increase, extend counter-selection as one sees fit and select the incubation time of cell and DNA library so that counter-selection choosing carry out more completely so that literary
The poor DNA removal of specificity is more thorough in library；It reduces the dosage of DNA library simultaneously and is incubated for it with positive sieve cell
Time shortens, and washing intensity increases.The screening efficiency of aptamers not only can be improved in this way, moreover it is possible to obtain screening suitable
Ligand has more preferably binding ability.It is as shown in table 1 to the specific detail of the regulation of screening pressure herein.
1 screening pressure of table regulates and controls detail list
| Number of screening round | Library dosage (pmol) | Positive sieve culture dish specification (mm) | Positive sieve incubation time (min) | Counter-selection culture dish specification (mm) | Counter-selection incubation time (min) | Washing buffer volume (mL) |
| 1 | 5000 | 10 | 60 | - | - | 2 |
| 2 | 278 | 10 | 60 | - | - | 2 |
| 3 | 289 | 10 | 55 | - | - | 2 |
| 4 | 345 | 10 | 55 | 6 | 30 | 2 |
| 5 | 336 | 10 | 50 | 6 | 30 | 3 |
| 6 | 356 | 10 | 50 | 6 | 35 | 3 |
| 7 | 340 | 6 | 45 | 6 | 35 | 3 |
| 8 | 320 | 6 | 45 | 6 | 40 | 3 |
| 9 | 300 | 6 | 40 | 6 | 40 | 4 |
| 10 | 280 | 6 | 40 | 10 | 45 | 4 |
| 11 | 260 | 6 | 35 | 10 | 45 | 4 |
| 12 | 240 | 6 | 35 | 10 | 50 | 4 |
| 13 | 220 | 6 | 30 | 10 | 50 | 5 |
| 14 | 200 | 6 | 30 | 10 | 55 | 5 |
| 15 | 200 | 6 | 30 | 10 | 55 | 5 |
| 16 | 200 | 6 | 30 | 10 | 60 | 5 |
| 17 | 200 | 6 | 30 | 10 | 60 | 5 |
| 18 | 200 | 6 | 30 | 10 | 60 | 5 |
| 19 | 200 | 6 | 30 | 10 | 60 | 5 |
4. sequencing result Primary Structure Analysis
After the completion of screening, the 19th wheel screening product is subjected to unmarked PCR amplification, being sent later to the raw work bioengineering in Shanghai has
Limit company carries out TA clone, selects 50 clones at random later and is sequenced.Company returns to ordered sequence 50, is all sequenced
Success.Compared with primary libraries, only 1 sequence random areas base quantity reduces 1, remaining all sequences with original text
Base quantity (84bp) of the library with same length, and by comparing discovery, all sequences design on having the same with original
Downstream fixed area.Primary structure is carried out to these sequencing results using Clustal X 2.0.3 software and compares analysis.By Fig. 3-
Shown in 6, acquired results are sequenced in addition to the complete repetitive sequence being mentioned above, also have between partial sequence with more similar
Base, as shown in Fig. 2.Analysis is compared as a result, sequencing result can be greatly classified into five families according to primary structure.It can be with
Find out, among these families, some family's more control sequences are that AT base contents are more abundant, the nucleic acid of these families
Sequence may have more flexible secondary structure, and have some family's more control sequences that then there is GC base more abundant to contain
Amount, these sequences may have more stable structure.Therefore it also needs using laboratory facilities such as flow cytometries to these families
Sequence is further investigated.
5. candidate aptamers fluorescent microscopic imaging
By verifying obtain three lesser candidate aptamers (PPP1-2, PPP1-4, PPP1-5) affected by environment respectively with target
Cell 293T-PPP1 and counter-selection select cell 293T to be incubated for, and combine situation to observe it using fluorescence microscope.As a result
As shown in figs. 3 and 4, after three candidate aptamers and target cell 293T-PPP1 are incubated for, cell membrane surface has stronger green glimmering
Light expression, wherein most bright with PPP1-4 fluorescence intensity；And after being incubated for control cell 293T, there is no green is glimmering for cell membrane surface
Light expression.It should be the results show that this three candidate aptamers have stronger binding ability, and most with PPP1-4 binding ability
By force, the results showed that, the present invention is using candidate aptamers PPP1-4 as high spot reviews object.
6. influence of the pancreatin to PPP1-4 binding ability
For further verify PPP1-4 binding site whether cell membrane surface protein, target cell is located in advance using pancreatin
Reason.As a result such as Fig. 5, illustrate that fluorescence of the target cell 293T- PPP1 in conjunction with candidate aptamers PPP1-4 is believed after digesting through pancreatin
Number there is a reducing tendency, and this reducing tendency lengthened and degree is aggravated with pancreatin action time.Pancreatin is processed as the result is shown
Cell surface protein destroyed, so that the combination of aptamers PPP1-4 and cell membrane surface protein is affected, to inhibit
The fluorescence signal intensity of aptamers PPP1-4 and cell combination.
7. sequencing result secondary structure analysis
Since aptamers function in conjunction with target cell, the secondary structure of DNA sequence dna is depended greatly on, therefore,
While investigating primary structure homology, the mould of secondary structure situation has been carried out to each family's DNA sequence dna using NUPACK
Quasi-, simulated environment selects combination buffer condition, i.e. monovalent cation concentration is 150 mM, and divalent cation concentration is 4 mM,
Temperature is 4 °C.The results show that the aptamers sequence of each family has the same or similar secondary structure mostly, therefore select
It is glimmering to carry out FITC as candidate aptamers for the higher aptamers sequence of representative or frequency of occurrence in each family
The candidate aptamers sequent synthesis of signal.Specific candidate's aptamers sequence is shown in Table 2.
The candidate aptamers sequence of table 2 and its equilibrium dissociation constant
| Title | Sequence (5 ' -3 ') | Kd (nM) |
| PPP1-1 | GAATTCAGTCGGACAGCGACGCAAGGATAGTAATTAGGTTTGGTGCGGTGGGGTAATTTCAGCGATGGACGAATATCGTCTCCC | 46.79 ± 27.15 |
| PPP1-2 | GAATTCAGTCGGACAGCGGAGAATGGCGTTAAGCTTTTCTTCCCTTGTGTTTGATTCTTAACCGATGGACGAATATCGTCTCCC | 81.99 ± 26.93 |
| PPP1-3 | GAATTCAGTCGGACAGCGTATTACAAGGCTCTCAGCCGAGCGGCCCCGGCTCCTAGGGGAATAGATGGACGAATATCGTCTCCC | 52.30 ± 46.78 |
| PPP1-4 | GAATTCAGTCGGACAGCGGATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTCGATGGACGAATATCGTCTCCC | 5.58 ± 3.11 |
| PPP1-5 | GAATTCAGTCGGACAGCGCAGTAATCCCTTGTGTTTGATGCTCATTTCTCTCTGAAATTGCTCGATGGACGAATATCGTCTCCC | 38.52 ± 19.72 |
The design of 8.PPP1-4 optimization
In conclusion the present invention screens the aptamers PPP1-4 obtained has strongest knot in numerous aptamers candidate sequences
Cooperation is used, therefore it can be expected that its in tumor associated target from now on to playing certain effect in Clinics and Practices.However, with first
Beginning library is identical, and PPP1-4 is the DNA sequence dna with 84bp length, and in various functional studies, DNA sequence dna length
Longer, stability is also poorer.Moreover, reasonably certain in removal aptamers sequence not function, even because of space
Occupy-place and influence aptamers play combination base sequence, it is most important to the acquisition of more excellent aptamers.Therefore, of the invention
Sequence optimisation further is carried out to PPP1-4, this optimization is main by the way of sequence truncation, i.e., by former sequence both ends primer
Carry out all or part of removal.Each aptamers sequence is as shown in table 3 after optimization truncates.
3 PPP1-4 of table optimizes truncated sequence
| Title | Sequence (5 ' -3 ') |
| PPP1-4 | GAATTCAGTCGGACAGCGGATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTCGATGGACGAATATCGTCTCCC |
| PPP1-4a | GATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTCGATGGACGAATATCGTCTCCC |
| PPP1-4b | GAATTCAGTCGGACAGCGGATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTC |
| PPP1-4c | GATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTC |
9.PPP1-4 optimization binding ability is investigated
Flow cytometry is carried out to PPP1-4 optimization, investigates the combination situation of itself and target cell 293T- PPP1.Knot
For fruit as shown in fig. 6, compared with former sequence, PPP1-4a and PPP1-4b have partial reduction for the binding ability of target cell, and
The binding ability of PPP1-4c and target cell is not substantially change.The result shows that the aptamers sequence that the present invention obtains is playing knot
When cooperation is used, may and disobey PPP1-4c also can play good combination with target cell 293T-PPP1.And with original
Sequence is compared, and truncated sequence PPP1-4c can improve to a certain extent stability and avoid the energy being degraded since length shortens
Power, thus more outstanding Targeting Effect may be played in follow-up study.
The foregoing is only a preferred embodiment of the present invention, and method involved in the present invention is to ATG16L1, CSNK2
The aptamer screening of equal tumor markers is equally applicable, is not intended to restrict the invention, for those skilled in the art
For, the invention may be variously modified and varied.It is any modification done within the spirit and principles of the present invention, equivalent
Replacement, improvement etc., should be included within scope of the invention.
Sequence table
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Claims (9)
1. a set of tumor-marker molecular nucleic acid aptamers based on CRISPR/Cas9, it is characterised in that: the aptamers are
One or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID.
2. the tumor-marker molecular nucleic acid aptamers of CRISPR/Cas9 according to claim 1, it is characterised in that: described
Aptamers have and be with the homology of one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID
60% or more sequence.
3. the tumor-marker molecular nucleic acid aptamers according to claim 1 based on CRISPR/Cas9, it is characterised in that:
The aptamers have to be hybridized with one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID
Sequence.
4. the tumor-marker molecular nucleic acid aptamers according to claim 1 based on CRISPR/Cas9, it is characterised in that:
The aptamers have is transcribed with one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID
Sequence.
5. the tumor-marker molecular nucleic acid aptamers according to claim 1 based on CRISPR/Cas9, it is characterised in that:
The aptamers have to be optimized with one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID
Truncated sequence.
6. the tumor-marker molecular nucleic acid aptamers according to claim 1 based on CRISPR/Cas9, it is characterised in that:
Several positions in the aptamers sequence are phosphorylated, methylate, amination, sulfhydrylation or isotopologue.
7. the tumor-marker molecular nucleic acid aptamers according to claim 1 based on CRISPR/Cas9, it is characterised in that:
Biotin, digoxin, fluorescent material, nano luminescent material, polyethylene glycol, peptide fragment, egg are combined in the aptamers sequence
White, folic acid or enzyme label.
8. the tumor-marker molecular nucleic acid aptamers according to claim 1 based on CRISPR/Cas9, it is characterised in that:
The tumor-marker molecular nucleic acid aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or
Several sequence alterations at corresponding peptide nucleic acid.
9. a set of tumor-marker molecular nucleic acid aptamers based on CRISPR/Cas9 described in claim 1-7 any one,
Be characterized in that: the aptamer is in the application for preparing tumor-marker molecular detection kit or detection reagent paper.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110161244A (en) * | 2019-05-10 | 2019-08-23 | 长沙医学院 | It is a kind of for detecting and the nucleic acid device and its construction method of modulate tumor mRNA |
| CN114807240A (en) * | 2021-01-21 | 2022-07-29 | 深圳市第二人民医院(深圳市转化医学研究院) | Aptamer-linked template molecule and kit thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343127A (en) * | 2013-07-24 | 2013-10-09 | 厦门大学 | Aptamer LXL-2 for breast cancer cell MDA-MB-231 and applications of aptamer |
| CN104364375A (en) * | 2012-06-04 | 2015-02-18 | 日本电气方案创新株式会社 | Nucleic acid molecule binding to influenza virus and use therefor |
| CN204330778U (en) * | 2014-09-22 | 2015-05-13 | 西南大学 | A kind of alkaline phosphatase detects immunity test strip fast |
| CN106645709A (en) * | 2016-09-30 | 2017-05-10 | 中国农业科学院农业质量标准与检测技术研究所 | Aptamer test strip adopting dual competitive method and application of aptamer detection strip |
| KR20180004503A (en) * | 2016-07-04 | 2018-01-12 | 부산대학교 산학협력단 | Selecting method of aptamer using AC potential modulated microfluidic channel |
| CN109456974A (en) * | 2018-07-02 | 2019-03-12 | 广西医科大学 | A kind of Endoglin aptamer and its application |
-
2018
- 2018-07-03 CN CN201810717217.2A patent/CN109576273A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104364375A (en) * | 2012-06-04 | 2015-02-18 | 日本电气方案创新株式会社 | Nucleic acid molecule binding to influenza virus and use therefor |
| CN103343127A (en) * | 2013-07-24 | 2013-10-09 | 厦门大学 | Aptamer LXL-2 for breast cancer cell MDA-MB-231 and applications of aptamer |
| CN204330778U (en) * | 2014-09-22 | 2015-05-13 | 西南大学 | A kind of alkaline phosphatase detects immunity test strip fast |
| KR20180004503A (en) * | 2016-07-04 | 2018-01-12 | 부산대학교 산학협력단 | Selecting method of aptamer using AC potential modulated microfluidic channel |
| CN106645709A (en) * | 2016-09-30 | 2017-05-10 | 中国农业科学院农业质量标准与检测技术研究所 | Aptamer test strip adopting dual competitive method and application of aptamer detection strip |
| CN109456974A (en) * | 2018-07-02 | 2019-03-12 | 广西医科大学 | A kind of Endoglin aptamer and its application |
Non-Patent Citations (5)
| Title |
|---|
| CELL—SELEX技术在肿瘤诊治中的应用进展: "吕彬等", 《复旦学报（医学版）》 * |
| YANLING SONG等: "Selection of DNA Aptamers against Epithelial Cell Adhesion Molecule for Cancer Cell Imaging and Circulating Tumor Cell Capture", 《ANALYTICAL CHEMISTRY》 * |
| 徐兴兴等: "PPP4R1 基因与肿瘤相关性的生物信息学预测及在胃癌组织中的初步验证", 《第二军医大学学报》 * |
| 许涛等: "核酸适配体筛选技术研究进展", 《生物技术通讯》 * |
| 赵永祥等: "基于DNA文库浓度递减的核酸适配体筛选方法及核酸适配体", 《科技成果》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110161244A (en) * | 2019-05-10 | 2019-08-23 | 长沙医学院 | It is a kind of for detecting and the nucleic acid device and its construction method of modulate tumor mRNA |
| CN110161244B (en) * | 2019-05-10 | 2022-02-18 | 长沙医学院 | Nucleic acid device for detecting and regulating tumor mRNA and construction method thereof |
| CN114807240A (en) * | 2021-01-21 | 2022-07-29 | 深圳市第二人民医院(深圳市转化医学研究院) | Aptamer-linked template molecule and kit thereof |
| CN114807240B (en) * | 2021-01-21 | 2024-02-06 | 深圳市第二人民医院(深圳市转化医学研究院) | Template molecule connected with aptamer and kit thereof |
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